| Objective Hepatic ischemia reperfusion injury (IRI) is a pathophysiologic process, which occured in many clinical courses, such as hemorrhage shock, toxic shock, liver transplantation, liver resction and so on. After hypoxia, the injury of blood reflow into organs has an important influence on the outcome of the shock/resuscitation and the liver graft survival rate after liver transplantation. Thus, the investigation of hepatic ischemia reperfusion injury will have important meaning to promote the recovery of the organ function after ischemia. Hepatic ischemia reperfusion injury is a complex multiple factor process. Oxygen free radidicals (OFR) formation, calcium overload, microcirculation disturbance were involved in the complex process. Heme oxygenase (HO) is an initial and limiting enzyme of heme oxidization, which can catalyze the heme into carbon monoxide (CO) , biliverdin and free iron. Recent studies found that HO and its metabolite take part in various pathophysiologic processes including resisting of the oxidative stress, regulating of the blood vessel tone, signal transduction, suppressing of proliferation in the cell and so on. Among these, HO expressed in the stress state can play a protective role on the tissues. Mitogen-activated protein kinase (MAPK) is an important signal transduction pathway related with oxidative stress. Among these, P38 play a key role in the state. The investigations of the relation between with P38 and hepatic ischemia reperfusion injury and between with P38 and HO-1 will help us to represent the pathogenisis. The present study was designed to investigate the role of heme oxygenase-carbon monoxide system in the pathogenesis of hepatic ischemia reperfusion injury in rats by using of heme oxygenase activator (hemin) and the selective heme oxygenase inhibitor (zine protoporphyrin, ZnPP). Method 1 Animal model and group The larotomy was performed in animal under anesthesia injected intraperitoneally with 10% chloral hydrate. Then, median incision was performed on the abdomen. The liver was exposed. After isolating carefully, the hepatic artery and portal vein of the liver left lobe and the middle lobe were clamped with miniclamp. Forty-five minutes later, the miniclamp was moved off. The model of hepatic ischemia reperfusion injury of the rats was performed. All male Wistar rats were divided into 4 groups at random: sham-operation group, ischemia reperfusion group, hemin group and ZnPP group. The hemin (20mg/kg) was injected intraperitoneally in the rats of hemin group at 12 hoursbefore ischemia reperfusive operation. The ZnPP (10umol/kg) was injected intraperitoneally twice at 3 hour and 16 hour before ischemia reperfusive operation in the rats of ZnPP group. 2 Mensuration of biochemical parameter The plasms of rats with ischemia reperfusive were collected at 0h, 4h, 8h and 12h after reperfusion. The levels of plasm alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined respectively by automatic biochemical analyzer. The homogenate of liver tissues of rats with ischemia reperfusive were preparated to determine the activities of superoxide dismutase (SOD) and malondialdehyde (MDA). 3 Liver histology analysis Paraffin sections were used. (1) Hematoxylin–Eosin (HE) staining. (2) Immunohistochemical staining: After deparaffinization and dehydration, sections were pretreated with 3% hydrogen peroxidase for 30min. Antigen heat plerosis: the slices were put into a volume filled with citric acid buffer, keeping the temperature of the buffer at 92~94℃for 15~25 min. After plerosis, the buffer was cooled down to room temperatue. Section was preincubated with goat serum at 37℃for 30min, then incubated with first antibody at 4℃for one night. The second antibody marked with biotin (1:100 dilution) was added into the section at 37℃for 30min, then dripping third antibody marked with horseradish enzyme at 37℃for 30min. Antigens were visualized with the 3,3-diaminobenzidine (DAB). At last,the slices were redyed with hematoxylin and closed over. |
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